Keratoconus is a bilateral ocular disorder that progressively thins and distorts the central portion of the cornea toward a conic shape, typically leading to substantial visual impairment and corneal scarring. Distortion of the cornea in keratoconus results from decreased resilience and low mechanical strength of the corneal tissues (Wollensak et al., Fortschr. Ophthalmol. 84:28-32, 1987, incorporated herein by reference). These structural defects represent important pathogenetic factors in the disease (Edmund, Acta Ophthalmol. 66:134-140, 1988, incorporated herein by reference), however the mechanisms that cause these structural changes remain undefined.
To date, no specific tools have been developed to treat or prevent keratoconus. In the mildest cases, management involves the use of spectacles or soft contact lenses. More commonly, early stage management of keratoconus requires specially designed contact lenses that compensate visual defects and provide some structural support to correct corneal distortion. More advanced presentations are managed with rigid gas-permeable (RGP) contact lenses to minimize corneal distortion and correct irregular astigmatism (Koliopoulos et al., Ann. Ophthalmol. 13(7):835-7, 1981, incorporated herein by reference). If satisfactory wearing time is not achieved with contact lens, or if the contact lens-corrected vision is not adequate (which may result from corneal scarring or poor fitting of the steeply sloped cone) keratoplasty is indicated.
Even with the aid of the foregoing management tools, the vision of patients with keratoconus often deteriorates beyond correction. At this point the replacement of corneal tissue by transplantation becomes the indicated treatment option. Corneal transplantation is necessary for 10% to 20% of patients with keratoconus (Kennedy et al., Am. J. Ophthalmol. 101(3):267-73, 1986; and Smiddy et al., Ophthalmology 95:487-92, 1988, each incorporated herein by reference). However, corneal transplantation is attended by high costs, limitation of the supply of suitable corneas, and substantial risks, including risks of adverse sequelae from anesthesia, transplant failure, and transmission of tissue-borne pathogens (e.g., HIV virus) from donor tissue to the transplant recipient.
In addition to visual defects, the medical history of keratoconus patients often elicits allergic or systemic conditions. Atopic disease (allergy) is present in approximately 35% of cases (Rahi et al., Br. J. Ophthalmol. 61:761-4, 1977, incorporated herein by reference). Eye rubbing, possibly a response to allergic discomfort, is reported by 20% of patients with keratoconus (Ridley, Br. J. Ophthalmol. 45:631, 1961, incorporated herein by reference). In addition, keratoconus has been associated with inherited systemic diseases such as Down syndrome, Lebers congenital amaurosis, osteogenesis imperfecta, and connective tissue disorders such as Ehier-Danlos syndrome.
Despite extensive clinical and research efforts, no definitive etiology has emerged for patients presenting with a diagnosis of keratoconus. Some evidence suggests that a genetic component is involved (Rabinowitz et al., Arch. Ophthalmol. 108:365-371, 1990; and Jacobs et al., Int. Ophthalmol. Clin. 33:249-260, 1993, each incorporated herein by reference). Other reports suggest that environmental conditions, such as excessive eye rubbing or contact lens wearing, contribute to the disease (Coyle, Am. J. Ophthalmol. 97:527-528, 1984; and Macsai et al., Arch. Ophthalmol. 108:534-538, 1990, each incorporated herein by reference). Yet additional causes have been proposed, including atopic disease and systemic conditions.
The prevalence of atopic disease, such as hay fever and asthma, is approximately three times more common for patients with keratoconus when compared with a matched control group (Rahi et al., Br. J. Ophthalmol. 61:761-4, 1977, incorporated herein by reference). Eye rubbing, perhaps a response related to the allergy, is also more frequently exhibited by patients with keratoconus (Ridley, Br. J. Ophthalmol. 45:631, 1961, incorporated herein by reference). However, a causal association between allergy and/or eye rubbing, and the onset or progression of keratoconus, has not been established.
Other reports in the literature suggest that rigid contact lens wear is a causal factor in some cases of keratoconus (Macsai et al., Arch. Ophthalmol. 108:534-8, 1990, incorporated herein by reference). This finding is difficult to substantiate, because keratoconus patients may self-select contact lens wear due to their refractive error or patient dissatisfaction with spectacles.
A variety of systemic conditions have also been associated with keratoconus. Exemplary conditions include connective tissue disease, such as Ehlers-Danlos Rieger""s, Crouzon""s, and Marfan""s syndromes. In addition, keratoconus has been diagnosed in up to 6% of patients with Down syndrome (Skeller et al., Acta. Ophthalmol. 29:149-61, 1951, incorporated herein by reference).
With regard to possible genetic factors, keratoconus has been shown to carry a hereditary component in approximately 6% to 8% of patients (Kennedy et al., Am. J. Ophthalmol. 101(3):267-73, 1986, incorporated herein by reference). In this context, Rabinowitz et al. (Arch. Ophthalmol. 108:365-71, 1990, incorporated herein by reference), have used videokeratographs to construct family pedigrees to determine modes of keratoconus heredity. The genetic component of keratoconus can be either dominant or recessive. However, the genotype of autosomal dominant is most common.
Despite the puzzling etiology of keratoconus, researchers continue to seek answers regarding the basic mechanisms underlying the onset and progression of this disease. However, to date most studies have focused on the morphological, i.e., histological and ultrastructural, changes associated with keratoconus. In this regard, it has long been noted that keratoconic corneas exhibit various ultrastructural defects, including fragmentation of Bowman""s layer, fragmentation of the epithelial cell basement membrane, and fibrillation of the anterior stroma (Teng, Am. J. Ophthalmol. 55:1847, 1963; Chi et al., Am. J. Ophthalmol. 42:847-60, 1956; and Bron et al., Trans. Ophthalmol. Soc. UK 98:393-6, 1978, each incorporated herein by reference). Possible causes of these defects have been suggested to include changes in the metabolism and composition of extracellular matrix materials (ECMs).
Bowman""s layer is an acellular matrix at the interface between the corneal epithelium and the stroma. It links the epithelial basement membrane and the stroma proper and may be crucial for epithelial attachment and function. During human corneal epithelial development, a distinct Bowman""s layer is formed at 19 weeks (Tisdale et al., Invest. Ophthalmol. Vis. Sci. 29:727-736, 1988, incorporated herein by reference). After birth, the thickness of Bowman""s layer remains unchanged. Components of Bowman""s layer are believed to be synthesized by both corneal epithelial and stromal cells, and an epithelial-stromal interaction is suggested to be a major factor in the formation of Bowman""s layer (Hay, Int. Rev. Cytol. 63:263-322, 1980, incorporated herein by reference).
Several years after radial keratotomy in human corneas, a Bowman""s layer-like structure is formed underneath epithelial plugs that extend into the stroma (Melles et al., Arch. Ophthalmol. 113:1124-1130, 1995, incorporated herein by reference). The collagen fibrils in Bowman""s layer are of relatively small diameter and are randomly arranged. In the underlying corneal stroma, the resident stromal cells are responsible for the maintenance and organization of the collagens. However, considering that Bowman""s layer is acellular, the organization and maintenance of collagens at this site remains unexplained. One possibility is that these functions are performed by the sparse stromal cells that transverse into Bowman""s layer. Cytokines have also been suggested to play a role in collagen maintenance in Bowman""s layer. In keratoconus, the collagen maintenance function of stromal cells and/or cytokines may be disturbed for both Bowman""s layer and for the stroma.
For more than a decade, evidence has been accumulating to suggest that biochemical and/or molecular changes in the metabolism and/or makeup of extracellular matrix materials (ECMs), such as collagen, may lead to the above noted corneal structural changes associated with keratoconus. To evaluate this aspect of the disease, one comparative study analyzed collagen types I, III, IV and V in normal versus keratoconic corneas (see, e.g., Oxlund et al., Acta. Ophthalmol. 63:666-669, 1985, incorporated herein by reference). This study found no differences in amino acid composition, nor in the type and number of collagen cross-links, between these groups. Other studies reported changes in the spatial distribution and specific immunostaining of the various collagen types between normal and keratoconic corneas (Ihme et al., Exp. Eye Res. 36:625,631, 1983; Nakayasu et al., Ophthalmic Res. 18:1-10, 1986; Yue et al., Proc. Soc. Exp. Biol. Med. 175:336-341, 1984; and Zimmermann et al., Exp. Eye Res. 46:431-442, 1988, each incorporated herein by reference). However, one study reported increased content of type V collagen in keratoconic corneas (Yue et al., Biochim. Biophys. Acta. 755:318-325, 1983, incorporated herein by reference).
Other studies directed to protein metabolism and content in keratoconic corneas produced different results compared with the above cited studies. For example, some reports conclude that keratoconic tissues exhibit higher levels of protein and increased incorporation of protein precursors (e.g., [3H]-proline) into all cell layers compared with normal corneal tissues (Rehany et al., Invest. Ophthalmol. Vis. Sci. 25:1254-1257, 1984; Critchfield et al., Exp. Eye Res. 46:953-963, 1988; and Wollensak et al., Graefe""s Arch. Clin. Exp. Ophthalmol. 228:517-523, 1990, each incorporated herein by reference). In contrast, Sawaguchi et al. conclude that collagen content in some cases of keratoconus is reduced compared with that of normal human corneas (Arch. Ophthalmol. 116:62-68, 1998, incorporated herein by reference). These authors also report abnormal, loosely packed and randomly oriented collagen fibrils in the corneal stroma of some keratoconus patients, which is proposed to reflect reduced collagen density. Further complicating this model, Yue et al. report that corneal specimens and cultured cells from affected persons show a reduction in overall protein levels compared with normal controls (Proc. Soc. Exp. Biol. Med. 175:336-341, 1984, incorporated herein by reference). However, collagen content is normal in some specimens, while the levels of collagenous proteins is substantially reduced in others, suggesting that pathology of the disease""s is in fact xe2x80x9cheterogeneousxe2x80x9d.
Reports of reduced protein levels in keratoconic corneas have generated numerous hypotheses that the disease may be causally linked to changes in the degradative metabolism of extracellular macromolecules (Yue et al., Proc. Soc. Exp. Biol. Med. 175:336-341, 1985, incorporated herein by reference). In this context, researchers have proposed a variety of possible mechanisms, including changes in the structure, expression, and/or activity of degradative enzymes, and alterations in the levels and/or activity of enzyme inhibitors. Because interacting systems of degradative enzymes and their inhibitors are normally under tight regulatory controls, alterations in enzyme inhibitor balances may have a significant impact on the integrity of the cornea.
Studies aimed at defining degradative changes associated with keratoconus have yielded diverse, and often conflicting, results. A number of studies report increased levels of degradative enzymes associated with keratoconus (Sawaguchi et al., Arch. Ophthalmol. 107:1507-1510, 1989, incorporated herein by reference). For example, keratoconic corneas reportedly exhibit increased levels or activities of acid esterase, acid phosphatase, acid lipase, and cathepsins B and G (Sawaguchi et al., Invest. Ophthalmol. Vis. Sci. 35:4008-4014, 1994; Sawaguchi et al., Arch. Ophthalmol. 107:1507-1510, 1989; and Zhou et al., Invest. Ophthalmol. Vis. Sci. 39:1117-1124, 1998, each incorporated herein by reference). Also reported are higher collagenase and gelatinase activities in keratoconic tissues compared to normal corneal tissues (Kao et al., Biochem. Biophys. Res. Commun. 107:929-936, 1982, incorporated herein by reference). In light of these reports, some investigators propose that imbalances in protease/protease inhibitor levels may contribute to decreased protein levels and increased proteolytic activities associated with keratoconus. Additional investigations have reported decreases in the levels of certain proteolytic enzyme inhibitors, for example, xcex11-protease inhibitor (alp1) (Whitelock et al., Invest. Ophthalmol. Vis. Sci. 38:529-534, 1997; Sawaguchi et al., Exp. Eye Res. 50:549-554, 1990, each incorporated herein by reference), and xcex12-macroglobulin (xcex12-M) (see, e.g., Sawaguchi et al., Arch. Ophthalmol. 107:1507-1510, 1989; Sawaguchi et al., Exp. Eye. Res. 50:549-554, 1990; and Sawaguchi et al., Invest. Ophthalmol. Vis. Sci. 35:4008-4014, 1994, each incorporated herein by reference) in association with keratoconus.
However, the model of a protease/protease inhibitor imbalance as a pathogenic mechanism in keratoconus remains speculative. In this regard it is noteworthy that Yue et al., supra, conclude that, for the subset of keratoconus cases where collagen levels are actually reduced, the reduction is apparently due to decreased collagen synthesis rather than to an increase in protease activity or a decrease in protease inhibition. Other reports suggest that pathogenic changes associated with keratoconus, including increased collagenolytic activity, may be attributed to structural changes in the collagen proteins themselves, as opposed to altered collagen expression or elevated protease function (e.g., attributable to higher protease levels or activities, or decreased protease inhibitor levels or activities).
A substantial amount of research pertaining to degradative mechanisms in keratoconus has focused on a large class of proteolytic enzymes, known as matrix metalloproteases (MMPs). MMPs comprise a large class of enzymes known for their capacity to degrade extracellular matrix elements. This class includes a growing host of collagenases (e.g., MMP-1, MMP-8, MMP-13, and MMP-18), gelatinases (e.g., gelatinase A or MMP-2, and gelatinase B or MMP-9), and stromelysin (MMP-3). MMPs are generally secreted as proenzymes and must be activated for conversion into mature enzymes. MMP species and activities present in keratoconic corneas have been extensively examined.
Data obtained by assaying acyl transferase activity show that MMPs account for at least 95% of the total protease secreted by cultured keratocytes. The summated specific activity of MMPs is reported to be consistently and significantly higher in the culture media of keratoconic keratocytes than in media of other keratocyte cultures. Smith et al., Eye 9:429-433, 1995, incorporated herein by reference. More specifically, researchers have reported significantly increased collagenase and gelatinase activities associated with keratoconus, in both organ culture (Kao et al., Biochem. Biophys. Res. Commun. 107:929-936, 1982; and Rehany et al., Ann. Opthalmol.: 14:751-754, 1982, each incorporated herein by reference) and cell culture (Ihlainen et al., Eur. J. Clin. Invest. 16:78-84, 1984; and Kenney et al., Biochem. Biophys. Res. Commun. 161:353-357, 1989, each incorporated herein by reference) studies. However, at least one investigation using corneal extracts reported no difference between keratoconic and healthy control samples in either the total amount or types of gelatinases (Zhou et al., Invest. Opthalmol. Vis. Sci. 39:1117-1124, 1998, incorporated herein by reference). A subsequent study by Brown et al. reported that gelatinase activity was elevated in keratoconus extracts after chemical modification of inhibitory elements (Curr. Eye Res. 12: 571-8, 1993, incorporated herein by reference).
Although increased collagenase activity in keratoconus is expected to be associated with reduced collagen content in keratoconic corneas, widely varying results have been reported in this context. While some studies appear to demonstrate decreased collagen levels associated with the disease, others indicate that collagen levels remain unchanged. In addition, the results of Yue et al., supra, suggest that the disease is actually heterogeneous, with some cases associated with reduced collagen content and others not. These results militate against a causal relationship between collagen degradation and keratoconus. Specifically, because Yue et al. found no significant differences in medical histories and severity of clinical symptoms between the different groups studied, the observed variation in collagen content in one group of keratoconus patients does not appear to be directly linked to disease progression.
In situ zymography studies have also been conducted to further assess the net functional activity of MMPs in keratoconic tissues (Zhou et al., Invest. Opthalmol. Vis. Sci. 39:1117-1124, 1998, incorporated herein by reference). The results obtained from these studies reportedly showed that basal levels of gelatin and casein digesting activities were present in healthy human corneas, and that these activities were increased in keratoconus. Gelatin and casein are preferred substrates for gelatinases A and B, and stromelysin. They can, however, also serve as substrates for other proteases.
To determine whether gelatinolytic and caseinolytic activities associated with keratoconus are caused by MMPs or other classes of proteases, Zhou and coworkers (1998, supra) also employed inhibitors specific for four classes of proteases (aspartic, serine, cysteine, and metallo-proteases) as blocking reagents. These studies reportedly showed that, in both healthy controls and keratoconic specimens, the net gelatinolytic and caseinolytic activities were related mostly to serine and cysteine proteases, and not to aspartic proteases, gelatinases A and B, or stromelysin. The inhibitor of serine proteases phenylmethyl sulfonyl fluoride, the cysteine protease inhibitor E-64, and the cathepsin B-trypsin inhibitor leupeptin substantially reduced digestion of gelatin and casein, whereas the aspartic protease inhibitor pepstatin and the MMP inhibitor 1, 10-phenanthroline failed to block the reaction.
These in situ zymographic results may be contrasted with earlier organ culture and cell culture studies which suggested that increased gelatinase activities in keratoconus are caused by gelatinases A and B and stromelysin (Kat et al., Biochem. Biophys. Res. Commun. 107:929-36, 1982; Fini et al., Curr. Eye Res. 11:849-62, 1992; Ihalainen et al., Eur. J. Clin. Invest. 16:78-84, 1986; and Brown et al., Curr. Eye Res. 12: 571-8, 1993, each incorporated herein by reference). This model, which previously enjoyed wide acceptance in the keratoconus field, is rejected by Zhou and coworkers, partly on the proposed basis that MMPs may not be present in the cornea in active forms under nonpathologic conditions. In accordance with this model, Zhou and coworkers suggest that most gelatinolytic and caseinolytic activities in healthy human corneas may not be caused by MMPs. Instead, cysteine proteases, such as cathepsin B, and serine proteases, such as cathepsin G, both of which are reportedly elevated in keratoconus, are proposed to contribute to the enhanced gelatin- and casein-digesting activities associated with keratoconus.
Yet other models have been presented to explain the net increase of gelatinase activity in corneas with keratoconus. For example, Sawaguchi and coworkers (Invest. Ophthalmol. Vis. Sci. 35:4008-4014, 1994, incorporated herein by reference) propose that this increase may be attributable to decreased levels of tissue inhibitors of metalloprotease (TIMPs) (citing Brown et al., Curr. Eye Res. 12:571-581, 1993, incorporated herein by reference) and a2-macroglobulin (citing Bouaboula et al., J. Biol. Chem. 267:21830-21838, 1992, incorporated herein by reference). Evidence presented in these and other reports suggests that certain protease inhibitors are reduced in association with keratoconic disease. For example, a decrease in al-protease inhibitor levels were reported in both the epithelial and the stromal layers of keratoconus corneas based on immunostaining and dot blot assays (Sawaguchi et al., Exp. Eye. Res. 50:549-554, 1990, incorporated herein by reference). However, when these results were supplemented by computerized colorimetry, it was found that the abnormal inhibitor levels in the stromal layer was confined to the stromal lamellae, and no reduction in the inhibitor level actually occurred in the keratoconus stromal cells. Other evidence based on measurements of mRNA levels suggest that the xcex11-protease inhibitor may be downregulated in association with keratoconus (Whitelock et al., Invest. Opthalmol. Vis. Sci. 38:529-534, 1997, incorporated herein by reference).
Despite the uncertainties apparent from these reports, a number of patent disclosures purport to teach methods for treating corneal disease using protease inhibitors. Specifically, each of the following patents, U.S. Pat. No. 5,962,481, issued Oct. 5, 1999 to Levin et al., U.S. Pat. No. 5,929,097, issued Jul. 27, 1999 to Levin et al., U.S. Pat. No. 5,773,438, issued Jun. 30, 1998 to Levy et al., and U.S. Pat. No. 5,892,112, issued Apr. 6, 1999 to Levy et al., state that synthetic, small molecule, non-peptide protease inhibitors may be useful in treating keratoconus. However, these disclosures add nothing to the foregoing reports with respect to identifying the actual mechanisms that underlie keratoconus, or to clearly elucidate potential treatment agents and modalities for clinical use against the disease. Moreover, each of these disclosures proposes that vast numbers of synthetic protease inhibitors may be used to treat a laundry list of diseases. Thus, both of the Levin et al. patents identify hundreds of synthetic protease inhibitors and generally assert that the disclosed compounds can be used to treat such diverse diseases and conditions as arthritis, tumor growth and metastasis, angiogenesis, tissue ulceration, abnormal wound healing, periodontal disease, bone disease, proteinuria, aneurysmal aortic disease, degenerative cartilage loss following traumatic joint injury, demyelinating diseases of the nervous system, graft rejection, cachexia, anorexia, inflammation, fever, insulin resistance, septic shock, congestive heart failure, inflammatory disease of the central nervous system, inflammatory bowel disease, HIV infection, age related macular degeneration, diabetic retinopathy, proliferative vitreoretinopathy, retinopathy of prematurity, ocular inflammation, keratoconus, Sjogren""s syndrome, myopia, ocular tumor, ocular angiogenesis/neovascularization. Most notably, none of the foregoing patents directed toward production and use of small molecule protease inhibitors for disease treatment provide specific direction or guidance as to the underlying mechanisms of keratoconus and other corneal diseases, nor to the preparation and use of effective agents and formulations for treating these diseases.
Further discussion regarding the possible roles of proteases and their inhibitors in keratoconus is also provided by Kenney et al. (Biochem. Biophys. Res. Comm. 161:353-357, 1989, incorporated herein by reference). This report focuses on a specific gelatinase, type IV collagenase, which shows marked preference for gelatin (denatured collagen), types IV, V, and VII collagen, and fibronectin as substrates. Notably, the corresponding progelatinase molecule demonstrates considerable amino acid similarity to human procollagenase and prostromelysin. However, regulation of these enzymes is independent, as indicated by studies demonstrating selective increases in progelatinase synthesis accompanied by decreased procollagenase production in response to transforming growth factor-xcex2 (Overall et al., J. Biol. Chem. 264:1860-1869, 1989, incorporated herein by reference). Also, unlike collagenase and stromelysin, levels of gelatinase expression are not enhanced by exposure of cells to tumor promoter 12-0-tetradecanolyphorbol 13-acetate, but are increased by transformation with H-ras oncogene and treatment with transforming growth factor-xcex2.
Kenney and coworkers report a qualitative disparity between normal and keratoconic tissue samples with regard to this gelatinase. Normal keratocyte culture media contains a greater quantity of this gelatinase than media from keratoconus cultures. However, the keratoconus media displays increased gelatinolytic activity. Further characterization showed that the gelatinolytic activity is inactivated by chelators, dithiothreitol (DTT) and B-mercaptoethanol but is not effected by lmM phenylmethanesulfonyl fluoride (PMSF).
Based on these results, Kenney et al. propose an alternative mechanism to the protease/protease inhibitor imbalance model proposed by others. In particular, the authors suggest that there may be an inherent difference between the diseased and normal gelatinase enzyme, for example due to post-transnational modifications, amino acid differences leading to increased susceptibility to activation, or other factors. This proposal is in general agreement with a model advanced by Rehaney et al., who suggest that there is a high level of collagenase in an active form in keratoconic corneas, without trypsin activation, whereas the corresponding enzyme(s) in normal corneas is present in a latent form (Ann. Opthalmol. 14:751-754, 1982, incorporated herein by reference).
Other studies directed toward identifying protein degradative mechanisms in keratoconus have focused on lysosomal enzyme abnormalities associated with the disease. Lysosomal enzymes are widely distributed in ocular tissues, including the cornea, and have been implicated in several ocular diseases, such as uveitis and retinal degeneration. In addition to these pathogenic associations, Sawaguchi et al. reported that corneas from patients with keratoconus exhibited elevated levels of three acid hydrolases when compared with normal human corneas (Arch. Opthalmol. 107:1507-1510, 1989, incorporated herein by reference). This elevation was most prominently seen with acid phosphatase and was highlighted in the basal epithelium of keratoconus specimens.
Lysosomal enzymes are active elements participating in the degradation of protein, polysaccharide, nucleic acids, and lipids into low-molecular-weight constituents under acidic pH. They are also involved, physiologically, in phagocytosis and catabolism, and, pathologically, in inflammation, immune responses, and lysosomal storage diseases. The finding that corneas with keratoconus contain higher-than-normal levels of lysosomal hydrolases thus provides yet another candidate for a molecular/biochemical determinant in keratoconus. However, further investigation is also required to determine whether this abnormality represents a primary, or incidental, factor in keratoconus disease development. Underscoring this point, Sawaguchi and colleagues reported significant heterogeneity in lysosomal enzyme levels among keratoconus specimens analyzed in their study. These variations could not be correlated to differences in medical histories, clinical features, or histopathologic characteristics, such as degree of scarring or keratometric readings, again pointing to a heterogeneous pathology for the disease.
Thus, as in the case of other postulated determinants of keratoconus, no conclusive evidence has emerged to implicate changes in ECM protein structure, expression, and/or degradative metabolism as pathogenetic causes in the disease. Reported changes in protease and protease inhibitor properties (e.g., expression, structure, activity or metabolism) are difficult to interpret, and to reconcile with other reports. Thus, it is unclear whether these changes may contribute to onset or progression of keratoconus, or merely represent incidental sequelae of the disease. Resolution of these alternative possibilities is further complicated by the possibility that protease and protease inhibitor activities may be altered in keratoconus as a result of concurrent wound-healing and repair mechanisms, allergic responses, or responses to mechanical trauma, rather than as a direct effect of the primary disease. For example, declines in the protease inhibitor levels in the cornea may simply be a reflection of a similar decline in tears, or in serum. It has been shown that hormonal balance (Findlay et al., Endocrinology 108:2129-2135, 1981) and allergic disease (Berman et al., Invest. Opthalmol. Vis. Sci. 12:759-770, 1973, each incorporated herein by reference) can affect protease inhibitor levels in tears and in serum, and allergic diseases are often associated with keratoconus (Rahi et al., J. Opthalmol. 61:761-764, 1977, incorporated herein by reference).
Still other biochemical and molecular mechanisms have been postulated to play a determinative role in the onset and progression of keratoconus. In particular, detailed studies concerning the role of extracellular matrix materials (ECMS) in keratoconus have focused on aberrant macromolecular and biochemical properties of native proteoglycans. The mechanical strength of the corneal collagen-fiber network depends not only on the formation of covalent cross-links between collagen molecules, but also on a precise interaction of collagen with matrix proteoglycans. These interactions stabilize the collagen network and maintain regular spacing of the collagen fibers. Thus, even minor deviations in the structure of proteoglycans may impair the stabilizing effect of proteoglycans on the collagen network, resulting in increased distensibility of the corneal tissue over time (Edmund, Acta. Ophthalmol. 65:545-550, 1987, incorporated herein by reference). This proposed mechanism is consistent with the slowly progressive development of keratoconus which often requires several years for full expression.
Consistent with the above noted histologic and ultrastructural findings, keratoconic corneas have been shown to exhibit abnormal accumulations of chondroitin- and dermatan sulfate-type proteoglycans around collagen fibrils and collagen lamellae (Sawaguchi et al., Invest. Ophthalmol. Vis. Sci. 32:1846-1853, 1991, incorporated herein by reference). In addition, numerous pores noted in the stroma of keratoconic corneas are thought to represent areas occupied by keratocytes and abnormal proteoglycan molecules (Sawaguchi et al., Arch. Ophthalmol. 116:62-68, 1998, incorporated herein by reference). These and other findings point to an association between keratoconic structural defects and aberrant properties of proteoglycans in the corneal matrix.
In one study directed to the role of ECM proteoglycans in keratoconus, Budeecke and Wollensak isolated ECM components from metabolically labeled, normal and keratoconic, human corneas (Graefe""s Arch. Clin. Exp. Ophthalmol. 171:105-120, 1966, incorporated herein by reference). These studies revealed a significant increase in hexosamine content in the keratoconic cornea. No change was observed in the ratio of chondroitin sulfate/keratan sulfate (CS/KS) in keratoconic compared to normal corneas. Analysis of the overall mass and relative molecular mass (Mr) of the protein core and the glycosaminoglycan side chains of proteochondroitin sulfate (CS-PG) dermatan sulfate proteoglycan (DS-PG) and keratan sulfate-containing proteoglycan (KS-PG) revealed differences between normal and keratoconic cornea in the ratio of DS-PG/KS-PG, and in the chain length of KS chains. The latter finding suggests a higher proportion of KS-PG molecules having a lower than normal molecular weight, corresponding to a reduction of about 40% in the length of KS chains, in keratoconic compared to normal corneas.
Further suggesting a causal role for extracellular matrix glycoproteins in keratoconus are reports documenting marked increases in uronic acid, neutral hexoses, and N-acetylgalactosamine as well as elevated amounts of corneal glycoconjugates in keratoconus extracts compared to normal controls (Critchfield et al., Exp. Eye Res. 34:83-98, 1982; Yue et al., Arch. Ophthalmol. 106:1709-1712, 1988, each incorporated herein by reference). Based on these and other reports, researchers have proposed that the structure, composition and/or levels of ECM glycoproteins in the cornea may be important determinants of the onset and progression of keratoconus.
The role of proteoglycan structure and metabolism in keratoconus may be influenced by a variety of complex molecular and biochemical factors. In this context, recent discussion has been directed toward specific proteases as potential modulators of proteoglycan structure/metabolism. Cathepsin B, a cysteine protease, and cathepsin G, a neutral serine protease, are both enzymes known to degrade proteoglycans in the corneal stroma, which is the site of thinning and scarring in keratoconus. Both are synthesized as large-molecular weight precursors containing signal- and pro-sequences that undergo several post-transcriptional modifications before being targeted to lysosomes and possibly other compartments of the endocytic or secretory pathways (Wang et al., J. Biol. Chem. 266:12633-12638, 1991, incorporated herein by reference). In human colorectal carcinoma, (Jessup et al., Am. J. Pathol. 145:1-11, 1994, incorporated herein by reference) and during fetal calf myoblast-myotube differentiation, (Bechet et al., J. Biol. Chem. 266:14104-14112, 1991, incorporated herein by reference), the activity of cathepsin B has been shown to be increased due to induction of cathepsin gene transcription, alteration in the secretion pathway, or modification of enzyme properties. The increased cathepsin B and G activities in keratoconus also may be related to increased gene transcription (Whitelock et al., Invest. Ophthalmol. Vis. Sci. 38:529-534, 1997, incorporated herein by reference). However, at present, the exact mechanisms by which cathepsin expression is controlled in the cornea under nonpathologic conditions, and how those conditions are altered in association with keratoconus, remain poorly understood.
Although the foregoing models embrace much of the current knowledge pertaining to biochemical and molecular mechanisms in keratoconus, new models for this disease continue to emerge. One such model focuses on aberrant epithelial-stromal interactions, which are thought to comprise a fumdamental aspect of disease development in keratoconus. In this regard, Wilson and colleagues have postulated that interleukin 1 (IL-1) may be a cytokine modulator of epithelial-stromal interactions, regulating corneal cell proliferation, differentiation, and cell death (Exp. Eye Res. 62:325-337, 1996, incorporated herein by reference). These investigators have also proposed a causal role of the IL-1 system in keratoconus. Support for this model includes findings that cultured keratoconus stromal cells contain 4-fold higher binding sites for IL-1 (Fabre et al., Curr. Eye Res. 7:585-592, 1991, incorporated herein by reference). Also noteworthy are studies which show enhanced expression of the IL-1 receptor associated with keratoconus. Zhou et al., Invest. Ophthalmol. Vis. Sci. 37(suppl):S1017, 1996, incorporated herein by reference. The hypothesis that IL-1 plays a causal role in keratoconus is also consistent with a model of corneal degradation mediated by matrix metalloproteases, because IL-1 is known to regulate the expression of MMPs in the cornea. Girard et al., Invest. Ophthalmol. Vis. Sci. 32:2441-2454, 1991, incorporated herein by reference.
Considering all of the above reports in their entirety, it is clear that the etiology of keratoconus remains uncertain. Whereas the gross anatomical, histological, and ultrastructural defects associated with keratoconus clearly represent proximate causes of the disease, the underlying biochemical and molecular mechanisms that trigger the onset and progression of these defects remain unclear and open to much debate. Most importantly, despite extensive efforts in the clinic and laboratory to model the disease and develop preventive and therapeutic tools, management of keratoconus generally remains limited to physical intervention by corrective lenses and transplantation surgery.
Information that is available concerning the ultimate, biochemical and molecular causes of keratoconus provides limited promise of future treatments or cures for the disease. In this context, the reports summarized above present complex and often conflicting etiologic models. Even simple genetic models for the disease are complicated by, or conflict with, models that embrace mechanical factors (e.g., eye rubbing or contact lens wear) as causes for the disease. Yet additional environmental causes, including atopic disease and systemic conditions, further complicate etiological modeling of the disease and support a heterogenous pathology attributable to a number of causes that can lead independently to common symptoms (see, e.g., Yue et al., Proc. Soc. Exp. Biol. Med. 175:336-341, 1984, incorporated herein by reference).
Although particular interest has been directed to ultrastructural defects in keratoconus (fragmentation of Bowman""s layer and epithelial basement membrane, and fibrillation of anterior stroma), no definitive mechanisms have been resolved to account for these changes. However, much attention has been directed to the extracellular Bowman""s layer, which resides at a dynamic interface between the corneal epithelium and stroma. Components of Bowman""s layer are thought to be synthesized by both corneal epithelial and stromal cells, and maintenance of this layer is believed to require complex epithelial-stromal interactions.
As noted above, it has been widely proposed that changes in the metabolism and/or composition of extracellular matrix materials (ECMs) are responsible for the observed defects in Bowman""s layer and other structural changes associated with keratoconus. However, the number and diversity of ECM components postulated to play a role in this context are extensive, and the complexity of proposed structural and metabolic interactions among these components is commensurately difficult to resolve.
Briefly summarizing the possible roles of ECM components in keratoconus, a large number of studies have focused on the role of collagen maintenance in the cornea. In this context, it has been proposed that the collagen maintenance function of stromal cells and/or cytokines may be disturbed for both Bowman""s layer and for the stroma. However, the organization and maintenance of collagens in Bowman""s layer remains unexplained.
Some studies that have examined the role of collagen maintenance in the cornea report no differences in spatial distribution and specific immunostaining, nor in amino acid composition and cross-linkages of collagens, between keratoconic and normal tissues. Other studies report abnormal, loosely packed and randomly oriented collagen fibrils in the corneal stroma of some keratoconus patients. Yet additional studies conclude that collagen content is increased, while others report a reduction in collagen content associated with keratoconus.
Further complicating the prospective etiology of keratoconus are reported changes in the proteoglycan content and metabolism that attend this disease. In particular, keratoconic corneas exhibit abnormal accumulations of chondroitin- and dermatan sulfate-type proteoglycans around collagen fibrils and collagen lamellae. In addition, there is a significant increase in hexosamine content in keratoconic corneas. Of particular note are changes in the ratio of dermatan sulfate proteoglycan (DS-PG) and keratan sulfate-containing proteoglycan (KS-PG) between normal and keratoconic corneas, as well as alterations in the length of KS chains. Further suggesting a causal role for extracellular matrix glycoproteins in keratoconus are noted increases in uronic acid, neutral hexoses, and N-acetylgalactosamine as well as elevated amounts of corneal glycoconjugates associated with the disease
In summary, reports of altered protein and glycoprotein levels and metabolism in keratoconus have prompted researchers to speculate on numerous possible mechanisms that may underlie the disease. These postulated mechanisms variously involve one or more proposed alterations in the levels, structure, expression and/or metabolism of extracellular proteins and/or proteoglycans. These alterations may be independent from, or coupled with, changes in the levels, structure, expression and/or activity of degradative enzymes, which are in turn variously proposed to be independent from, or associated with, alterations in the levels, structure, expression and/or activity of enzyme inhibitors. As discussed in detail above, all of these proposed mechanisms remain unresolved with respect to their causal, or incidental, roles in the pathogenic processes of keratoconus.
In view of the foregoing, the underlying biochemical and molecular mechanisms that control the onset and progression of keratoconus have heretofore remained unclarified. Thus, prior to the instant invention, an adequate platform from which to begin developing effective tools to treat or prevent this disease has remained out of reach.
It is therefore an object of the invention to provide effective therapeutic methods and compositions to treat and prevent keratoconus and other corneal disorders sharing common etiological characteristics of aberrantly high proteolytic activity with keratoconus. Included among these additional disorders are pathogenic infections, ulcers, and responses to injury attended by aberrant proteolytic activity.
It is a fuirther object of the invention to achieve the foregoing objects within methods and compositions that are easy to administer and which employ formulations that optimize delivery of therapeutic agents to ocular target sites including extracomeal fluid (tears, aqueous humor, or vitreous humor), corneal tissues, and vitreous humor.
Consistent with the foregoing objects, it is an additional object of the invention to provide methods and compositions that minimize discomfort and other adverse sequelae (e.g., corneal trauma, chemical irritation, visual impairment, etc.) associated with the foregoing treatment methods and compositions.
It is yet another object of the invention to provide the foregoing treatment methods and compositions in a combinatorial manner with other therapeutic methods and agents, to alleviate keratoconus concurrently with other, secondary ocular disorders. Targeted secondary disorders in this context include, but are not limited to, pathogenic infection, eye discomfort or irritation, atopic disease, and visual impairment. In more detailed aspects, methods and compositions for treating keratoconus are adapted for combinatorial use with corrective contact lenses, including rigid gas-permeable (RGP) lenses.
The invention fulfills these objects and satisfies other objects and advantages by providing novel methods and compositions for the treatment or prevention of corneal disease in a mammalian patient.
The methods of the invention involve ocular administration of an antiproteolytic effective amount of a protease inhibitor in an opthalmically acceptable carrier. The protease inhibitor is preferably a protein or peptide protease inhibitor selected from an aspartic, serine, cysteine, or metallo-protease inhibitor, which may be derived from a natural source or produced in a native or modified form by recombinant or synthetic techniques known in the art. In most instances, recombinant or synthetic protease inhibitors are preferred, as these materials will generally be free of undesirable contaminants and infectious agents.
According to the methods of the invention, a protease inhibitor formulation is administered to an ocular fluid, surface, or tissue, preferably by topical administration, in an antiproteolytic effective amount to substantially inhibit a proteolytic activity associated with the corneal disease or condition to be treated. Proteolytic activities in this context may include activities of multispecific or specific proteases, complex formation between a protease and protease inhibitor, histopathological changes in the cornea attributed to proteolytic processes, and other indicia correlated with proteolytic mechanisms.
The antiproteolytic formulations of the invention can include various carriers that prolong retention and/or enhance delivery of the inhibitor. Optionally, the antiproteolytic formulations can include permeabilizing agents and preservatives, which may be a single agent that enhances permeability and provides a simultaneous preservative function. In addition, the formulation can include a plurality of protease inhibitors, as well as other therapeutic agents such as antiinflammatory or antibiotic drugs.
Mode, timing, and duration of treatment according to the methods of the invention vary in accordance with a variety of factors detailed below. In preferred aspects of the invention, antiproteolytic formulations are administered during periods when contact lens are worn and/or of closed eye tear production to enhance therapeutic efficacy.
Also provided within the invention are implant devices adapted for corneal delivery of an effective amount of an antiprotease. These devices are provided in the form of an ocular implant having a concave inner surface similar in size and shape to the inner surface of a contact lens. The device is applied externally to the cornea of a patient suffering from a corneal disease or condition and serves as a carrier to deliver to the cornea an antiproteolytically effective amount of a protease inhibitor.
The instant invention provides useful methods and compositions for treating or preventing corneal disease in a mammalian patient. The methods of the invention involve ocular administration of an antiproteolytic effective amount of a protease inhibitor in an opthalmically acceptable carrier. The protease inhibitor is preferably selected from an aspartic, serine, cysteine, or metallo-protease inhibitor, obtained from a natural source or produced in a native (i.e., wild-type amino acid sequence) or modified (e.g., by amino acid substitution, insertion, deletion, truncation or extension, or by activation, fusion or conjugation with other proteins or chemical moieties) by recombinant or synthetic techniques known in the art.
According to the methods of the invention, a protease inhibitor formulation is administered to an ocular fluid, surface, or tissue, preferably by topical administration, in an antiproteolytic amount effective to substantially inhibit a proteolytic activity associated with the corneal disease or condition to be treated.
By substantial inhibition of proteolytic activity is meant that administration of the protease inhibitor formulation yields at least about a 10% reduction of proteolytic activity, preferably at least a 20% reduction, compared to a relevant baseline or control value at an ocular target site, for example within the extracorneal fluid, corneal epithelium, corneal stroma, Bowman""s layer, or the vitreous humor. Preferably, administration of the protease inhibitor yields approximately a 30-50% reduction of proteolytic activity, more preferably greater than about a 50% reduction, and in some preferred aspects yields effective neutralization of proteolytic activity in a treated sample corresponding to a reduction of between approximately 85% and 100% of the proteolytic activity measured for the relevant baseline or control sample.
As used herein, proteolytic activity refers to a quantitative digestive activity of a target protease against a protein (e.g., collagen, elastin, fibronectin) or glycoprotein (e.g., a proteoglycan or glycosaminoglycan) substrate. Target proteases as herein defined include proteolytic enzymes that exhibit aberrantly high levels of expression or activity (e.g., attributable to structural changes that increase substrate binding or otherwise enhance digestion kinetics, or that render the protease more susceptible to activation from a proenzyme to an active form), or whose regulation (e.g., by metabolic turnover, protease inhibition or other mechanisms) is impaired in association with a corneal disease or condition to be treated, for example keratoconus or corneal infections. As further defined herein, target proteases are amenable to regulatory inhibition by exogenously administered protease inhibitors.
Proteases that may be successfully targeted for inhibition by the compositions and methods of the invention include, but are not limited to, acid esterases, acid phosphatases, acid lipases, cathepsins (e.g., cathepsin B and G), collagenases, elastases, tryptases, chymases, kinins, kalikreins, tumor necrosis factors, chymotrypsins, stromelysins, and matrix metalloproteases (e.g., gelatinase A or MMP-2, gelatinase B or MMP9, MMP1, MMP 8, and MMP 13).
Antiproteolytic activity may be determined by, e.g., various quantitative, in vitro or in vivo assays, for example by enzymatic and/or immunological assays using extracorneal fluid samples, samples from keratocyte culture media, or corneal tissue samples, as described herein below and as otherwise known in the art. Alternatively, antiproteolytic activity may be determined by other indicia, for example by quantitative changes in morphological or ultrastructural features attributable to proteolytic activity that are amenable to prevention or inhibition using the compositions and methods of the invention. Exemplary indicia in this context include quantitative changes in the extent of fragmentation of Bowman""s layer, fragmentation of the epithelial cell basement membrane, and/or fibrillation of the anterior corneal stroma. These indicia can be readily compared between treated samples and relevant control samples, for example by histopathological computer-aided image analysis that resolves percentages of optical field areas occupied by proteolytically altered versus normal histological structures (e.g, fragmented versus non-fragmented areas of Bowman""s layer).
The extent of antiproteolytic activity elicited by the compositions and methods of the invention (i.e., for determining efficacy and calibrating dosages) can be determined by a variety of assays that compare relevant test and control samples, as detailed in the examples below. Suitable in vitro test and control samples include cultured, normal and keratoconic keratocytes, respectively, each treated with an antiprotease formulation of the invention. Using these samples, protease inhibition can be measured at selected time points, for example, by assaying target protease-inhibitor complex formation, rates or levels of protein digestion attributed to the target protease, morphological indicia as noted above, and other parameters consistent with the quantitative values sought.
Alternate quantitative inhibition assays can be conducted using, e.g., cultured keratocytes or corneal tissues taken from subjects with keratoconic and normal corneas to provide, respectively, test and control samples for in vitro assays. For quantitative determination of in vivo protease inhibition, test and control samples may include extracorneal fluid or corneal tissue samples taken from subjects (e.g., a human or non-human mammal such as a rabbit) following administration of a protease inhibitor formulation (test sample), and following administration of a placebo comprising, e.g., a selected carrier without the protease inhibitor (control sample). Often, test and control samples will be provided by bilateral administration of test and control treatments to an individual patient. Other suitable test and control samples will be determined by those skilled in the art according to the objectives and methods of the chosen assay, as exemplified herein below.
Protease inhibitors that are useful within the invention are any of the inhibitors, their analogs, recombinantly modified variants, proteolytically active fragments, derivatives, or salts, which can inhibit target proteases as defined above. Preferably, the inhibitor is a protein or peptide of sufficient molecular size for use within the formulations described herein that provide for enhanced absorption, retention and delivery of the inhibitor at a site of treatment. In various preferred embodiments, the protease inhibitor may be selected from an aspartic, serine, cysteine, or metallo-protease inhibitor. Useful inhibitors may be derived from a natural source or produced in a native or modified form by recombinant or synthetic techniques known in the art. In more detailed aspects of the invention, protease inhibitors bind with one or more proteases that exhibit increased levels of expression or activity, or aberrant regulation, leading to pathogenic protein or glycoprotein degradation and/or morphologic changes associated with a corneal disease or condition to be treated.
As noted above, preferred protease inhibitors include native or modified aspartic, serine, cysteine, or metallo-protease inhibitors. Exemplary inhibitors in this context include xcex11-antiprotease (alp1, formerly known as xcex11-antitrypsin), xcex12-macroglobulin (xcex12-M), secretory leucocyte protease inhibitor (SLP1, formerly known as mucus proteinase inhibitor and antileukoprotease), xcex21-antigellagenase, xcex12-antiplasmin, serine amylyoid A protein, al -antichymotrypsin (xcex11-Achy), cystatin C, inter-xcex1-trypsin inhibitor, elafm, elastinal, aprotinin, phenylmethyl sulfonyl fluoride, the cysteine protease inhibitor E-64, the cathepsin B-trypsin inhibitor leupeptin, and the metalloprotease inhibitors TIMP-1, TIMP-2, and 1, 10-phenanthroline.
A particularly preferred protease inhibitor for use within the compositions and methods of the invention is xcex12-macroglobulin. This inhibitor is a high-molecular-weight (718 kD), homotetrameric glycoprotein implicated as a regulator of degradation for certain extracellular matrix components and other macromolecules. Unlike many other protease inhibitors, xcex12-macroglobulin is not highly specific for a preferred target protease, and is not particularly fast acting. Instead, xcex12-macroglobulin inhibits proteases from all four major classes and is considered to be relatively slow in its activity. Consistent with these properties, the mechanism of action by xcex12-macroglobulin is also unique. When this protease inhibitor reacts with a target protease, proteolytic cleavage in the xe2x80x9cbait regionxe2x80x9d of the inhibitor occurs, leading to a conformational change and trapping of the protease. A covalent bond is then formed between the protease and xcex12-macroglobulin. The protease-inhibitor complex is ultimately cleared from the circulation by a receptor-mediated mechanism.
Another preferred protease inhibitor for use within the compositions and methods of the invention is xcex1-1 protease inhibitor (alp1), a major protease inhibitor in human plasma synthesized mainly by parenchymal liver cells. Alp1 is a glycoprotein of 53 kDa that forms a 1:1 complex with its target enzyme, leukocyte elastase. In addition to this primary target, alp1 inhibitor also inhibits chymotrypsin, cathepsin G, trypsin, plasmin, and thrombin. It is present in most body fluids, as well as in many tissues and cells. Alp1 has been demonstrated in all three layers of normal cornea as well as in the tears and aqueous humor.
As noted above, useful compositions within the invention include formulations of antiprotease salts, derivatives and complexes. As used herein, the term pharmaceutically acceptable salts, derivatives and complexes retain the desired biological activity of the corresponding native antiprotease, and exhibit minimal undesired toxicological effects. Nonlimiting examples of useful antiprotease salts are acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid; base addition salts formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with an organic cation formed from N,N-dibenzylethylene-diamine, Dglucosamine, ammonium, tetraethylammonium, or ethylenediamine; or combinations of acid and base addition salts.
Pharmaceutically acceptable derivatives and complexes of protease inhibitors include native or modified inhibitors that are chemically modified (e.g., by addition of stabilizing or otherwise finctional chemical moieties), truncated, conjugated (e.g., to a second protein, peptide or carrier) or recombinantly modified (e.g. by site directed mutagenesis using a cDNA encoding the inhibitor to introduce substitute, or delete non-critical amino acid residues), which retain desired biological activity of the corresponding native antiprotease.
Particularly useful in this context are protease inhibitor analogs, which comprise recombinantly modified variants and proteolytically active fragments of native inhibitors. These analogs preferably exhibit at least 80% amino acid identity, more preferably 95% or greater amino acid similarity, as compared to the amino acid sequence of the corresponding native inhibitor, as determined by conventional sequence alignment and comparison methods.
Alignment of amino acid sequences and calculation of percent identity between the aligned sequences is routine in the art. Such routine alignments include the introduction of gaps and employ other widely known conventions to account for sequence additions, deletions, conservative substitutions, etc. Briefly, conventional sequence comparison methods involve alignment of the compared sequences to yield the highest possible alignment score, which is readily calculated according to well known methods based on the number of amino acid or nucleotide matches.
Antiprotease analogs preferably share substantial amino acid sequence identity (e.g., at least 75%, preferably 80%, and more preferably 95% or greater sequence identity) with a xe2x80x9creference sequencexe2x80x9d of a corresponding native inhibitor protein or active polypeptide fragment thereof. As used herein, this reference sequence is a defined sequence used as a basis for a sequence comparison. Generally, a reference sequence is at least 20 amino acid residues in length, frequently at least 25 amino acid residues in length, and often at least 50 amino acid residues in length. Since analog and native fragment polypeptides may each (1) comprise a sequence (ie., a portion of the complete native sequence) that is similar between the two polypeptides, and (2) may further comprise a sequence that is divergent between the two polypeptides, sequence comparisons between two (or more) polypeptides are typically performed by comparing sequences of the two polypeptides over a xe2x80x9ccomparison windowxe2x80x9d to identify and compare local regions of sequence similarity. A xe2x80x9ccomparison windowxe2x80x9d, as used herein, refers to a conceptual segment of at least 20 contiguous amino acid residues wherein a polypeptide sequence may be compared to a reference sequence of at least 20 contiguous amino acid residues and wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman, (Adv. Appl. Math. 2:482, 1981, incorporated herein by reference), by the homology alignment algorithm of Needleman and Wunsch, (J. Mol. Biol. 48:443, 1970, incorporated herein by reference), by the search for similarity method of Pearson and Lipman, (Proc. Natl. Acad. Sci. USA 85:2444, 1988, incorporated by reference), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis., incorporated herein by reference), or by inspection, and the best alignment (i.e., resulting in the highest percentage of sequence similarity over the comparison window) generated by the various methods is selected. The term xe2x80x9csequence identityxe2x80x9d means that two polypeptide sequences are identical (i.e., on an amino acid-by-amino acid) over the window of comparison. The term xe2x80x9cpercentage of sequence identityxe2x80x9d is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (ie., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms xe2x80x9csubstantial identityxe2x80x9d as used herein denotes a characteristic of a polypeptide sequence, wherein the polypeptide comprises a sequence that has at least 80 percent sequence identity, preferably at least 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 amino acid residues, frequently over a window of at least 25-50 amino acid residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the analog sequence which may include modifications (e.g., deletions, substitutions, or additions) which total 20 percent or less of the reference sequence over the window of comparison. The reference sequence may be a subset of a larger sequence.
In addition to these polypeptide sequence relationships, protein analogs and peptide fragments of the invention are also typically selected to have conservative relationships with corresponding, native reference proteins and polypeptides. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Abbreviations for the twenty naturally occurring amino acids used herein follow conventional usage (Immunologyxe2x80x94A Synthesis (2nd ed., E. S. Golub and D. R. Gren, eds., Sinauer Associates, Sunderland, Mass., 1991), incorporated herein by reference). Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as xcex1,xcex1-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, xcex3-carboxyglutamate, xcex5-N,N,N-trimethyllysine, xcex5-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, xcfx89-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). Moreover, amino acids may be modified by glycosylation, phosphorylation and the like.
For practicing the methods of the invention, the precise amounts of protease inhibitors to be administered and the frequency and duration of treatment will depend on the status of the corneal condition or disease to be treated, and on other factors such as the patient""s state of health and weight, the mode of administration, the nature of the formulation, etc. These factors will vary such that specific regimens can be established by those skilled in the art to maximize efficacy of treatment. Ordinarily, the antiprotease is administered in a dosage of between approximately 0.2 xcexcg/ml and 1.0 mg/ml. Preferably, the inhibitor is present in a concentration of about 0.1-1.0 xcexcg/ml, more preferably at a concentration of about 0.5 xcexcg/ml. Exemplary formulations for xcex11-antitrypsin will comprise the inhibitor at approximately the same range of concentrations, with the most preferred concentration being between about 1.0 and 5.0 xcexcg/ml. The administration schedule can range from a continuous infusion, to once or twice a day, up to 6 or more administrations a day, with dose levels and administration protocols being selected by the health professional. Administration onto the concave surface of contact lenses before insertion into the eye is an effective method of enhancing the residence time for the solution in contact with the cornea.
Thus, treatments according to the invention can be in the form of a one time dose, e.g., in the context of sustained delivery and long-term delivery formulations described below. Alternatively, multiple administrations may be indicated and, under certain circumstances, continuous treatments may be selected.
In a preferred aspect of the invention, compositions comprising a protease inhibitor are administered during periods of closed eye tear production (e.g., during a patient""s sleep periods). This method greatly enhances antiproteolytic results, yielding prolonged inhibition of proteolytic processes in corneal tissues (e.g., as demonstrated by reduction in the activity of specific target protease(s)), and long-term inhibition of histopathological changes, such as fragmentation of Bowman""s layer. Administration of the antiprotease compositions of the invention during periods of closed eye tear production greatly enhances the antiproteolytic efficacy of these compositions compared to the efficacy achieved by antiprotease administration during periods of reflex tear production, although the latter use is effective and within the scope of the invention. This is due in part to the prolonged retention of the antiprotease composition attributable to a reduction in tear flushing between closed eye and reflex tear periods. This enhanced efficacy is also attributable to fundamental differences in the processes and regulation of proteolysis that characterize the closed eye, versus reflex tear environments. The protease inhibitor compositions and pharmaceutical formulations of the invention can also be administered during a period that is concurrent with or closely preceding a medical procedure or other event anticipated to produce a risk of proteolytic injury, for example following eye surgery or during bacterial infection. Thus, methods are provided which involve administration of an antiproteolytic composition concurrent with, or within an antiproteolytic effective period preceding or following, a surgical procedure or infection, whereby the administration reduces or eliminates risk of deleterious proteolytic responses normally associated with the procedure or infection.
Within the methods of the invention, formulations comprising a protease inhibitor, a mixture of a plurality of protease inhibitors, or a mixture of one or more protease inhibitors combined with a second therapeutic agent (e.g., an antibiotic, antiviral or antiinflammatory drug) can be administered by a variety of routes, including via topical administration (using, e.g., drops, gels, creams or microparticles as carriers), injection (e.g., via hypodermic or pneumatic introduction into the cornea or vitreous humor).
Preferred methods of the invention involve coordinate (e.g., simultaneous or closely contemporaneous to yield coordinate treatment) administration of a plurality of antiprotease proteins, analogs, salts, or derivatives, or administration of formulations comprising multiple protease inhibitors that may be admixed or complexed. Practice of these methods reduces abnormal proteolytic mechanisms attending a targeted corneal disorder (e.g., keratoconus) at combinatorial antiproteolytic levels that exceed antiproteolytic levels observed when either of the coordinately administered protease inhibitors are administered alone. This inhibition, as when other compositions and methods of the invention are employed, reduces proteolytic activity in extracorneal fluid (tears), and in corneal tissues (as determined by both enzymatic and histopathological assays). In preferred embodiments, a multispecific protease inhibitor (i.e., an inhibitor which targets multiple protease species), such as xcex12-M, SLP1 and alp1, is coordinately administered with another multispecific inhibitor, or, alternatively, a multispecific inhibitor is coordinately administered with an oligospecific or specific inhibitor (the latter types of inhibitors represented, e.g., by xcex21-antigellagenase, xcex12-antiplasmin, serine amylyoid A protein, xcex11-antichymotrypsin (xcex11-Achy), cystatin C, inter-a-trypsin inhibitor, elafin, elastinal, aprotinin, phenylmethyl sulfonyl fluoride, leupeptin, and the metalloprotease inhibitors TIMP-1, TIMP-2, and 1, 10-phenanthroline). Using these combinatorial compositions and treatment methods, the invention achieves effective inhibition against multiple proteases (and/or their pathogenic effects) involved in a particular corneal disease process. Thus, the methods and compositions of the invention provide antiproteolytic effects against a broad range of proteases, including but not limited to, acid esterases, acid phosphatases, acid lipases, cathepsins, collagenases, elastases, tryptases, chymases, kinins, kalikreins, tumor necrosis factors, chymotrypsins, stromelysins, and matrix metalloproteases, thereby alleviating or preventing the targeted corneal disease or condition.
Additional preferred methods of the invention involve coordinate administration of an antiprotease and an antibiotic, or administration of formulations comprising both a protease inhibitor and an antibiotic. Practice of these methods reduces abnormal proteolytic mechanisms attending a targeted corneal disorder (e.g., keratoconus) and also secondarily reduces proteolytic effects attributed to bacterial infection. Useful antibiotics may be any opthalmically acceptable antibiotic indicated for treatment of an ocular bacterial infection, including, but not limited to fluoroquinolones (e.g., ofloxacin, norfloxacin, ciprofloxacin), gentamicin, and pilocarpine.
Other medications useful in these combinatorial treatment methods include steroidal antiinflammatories, such as corticosteroids, and nonsteroidal antiinflammatories, for example aspirin, ibuprofen, indomethacin, fenoprofen, mefenamic acid, flufenamic acid, and sulindac. Many other combinatorially effective medicaments useful for coordinate ophthalmic treatment within the methods of the invention will be apparent to the skilled practitioner.
Typically, the protease inhibitors, analogs, salts, derivatives, and coordinately administered therapeutic agents of the invention will be administered in the form of a pharmaceutical composition, ie., dissolved or suspended in a physiologically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. Many other suitable carriers are known in the art and readily formulated with the subject therapeutic agents, including biologically compatible gels, creams, microparticulate solutions and the like suitable for topical administration. The pharmaceutical compositions may be sterilized by conventional, well known sterilization techniques. The resulting formulations may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution or other carrier prior to administration.
In other embodiments of the invention, the protease inhibitors, analogs, salts, derivatives, and coordinately administered therapeutic agents of the invention are prepared with carriers that protect the compound against rapid elimination from the ocular environment, such as are routinely used in controlled release devices and formulations (e.g., implants and microencapsulated delivery systems). Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, colla-gen, polyorthoesters, and polylactic acid. Such formulations and methods for their preparation will be apparent to those skilled in the art.
In a particularly preferred aspect of the invention, a novel delivery device is provided in the form of an ocular implant adapted for corneal delivery of an effective amount of an antiprotease. The device has a concave inner surface that conforms to an external surface of the cornea, i.e., which is similar in size and shape to the inner surface of a contact lens, and is applied externally to the cornea of a patient suffering from a corneal disease or condition. The device serves as a carrier to deliver to the cornea an antiproteolytically effective amount of a protease inhibitor. Preferably, the device is comprised of a gas-permeable, biocompatible polymer, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, or polylactic acid. The entire body, or at least an inner surface, of the device is coated or impregnated with the protease inhibitor. The device is disposable and provided in sterile packaging, to be implanted by the patient and worn for a selected treatment period, preferably for the full duration of a period of closed eye tear production (e.g., overnight) for maximum therapeutic efficacy.
Liposomal suspensions may provide useful, pharmaceutically acceptable carriers for formulating antiproteolytic compositions of the invention. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 (incorporated herein by reference). For example, liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the protease inhibitor is then introduced into the container. The container is swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.
The pharmaceutical compositions for use within the invention may also contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity-adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. The concentration of the antiprotease in these formulations can vary widely, i.e., from less than about 0.05%, usually at least about 0.5%, to as much as 15 or 20% by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
In preferred methods within the invention, mammalian subjects, including human patients, suffering from protease-mediated corneal disorders are treated by administering to the patient a pharmaceutical or therapeutic composition comprising an effective amount of one or more antiproteases, or a pharmaceutically acceptable derivative or complex thereof, in a pharmaceutically acceptable carrier or diluent.
The active antiprotease is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient an antiinflammatory effective amount without causing serious adverse side-effects in the patient treated. The active compound is preferably administered to achieve peak concentration of the antiprotease in tear fluid or corneal tissue of the patient within about 1-4 hours after administration. Concentration of the antiprotease in pharmaceutical compositions and devices of the invention will depend on such factors as absorption, distribution, inactivation, degradation, and flushing of the antiprotease, as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens may be adjusted over time according to the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the invention.
In view of the description and examples provided herein, the methods of the invention are shown to be useful for treating a wide variety of inflammatory conditions and diseases. In particular, the methods of the invention can be employed to treat keratoconus and other corneal disease or conditions characterized by aberrantly high proteolytic activity or damage attributable to aberrant proteolytic processes. Other conditions indicative of treatment using the compositions and methods of the invention include, but are not limited to, corneal ulcers, allergic conditions, bacterial infection, viral infection, corneal injury and post-surgical wound healing.
The pharmaceutical formulations administered within the methods of the invention must be opthalmically acceptable. In general, compounds and formulations with a therapeutic index of at least 2, preferably at least 5-10, more preferably greater than 10, are opthalmically acceptable. As used herein, the therapeutic index is defined as the EC50/IC50, wherein EC50 is the concentration of compound that provides 50% inhibition of a target proteolytic activity (e.g., proteolysis by a specific protease, or histopathologic change attributed to proteolysis) compared to a relevant control, and IC50 is the concentration of compound that is toxic to 50% of target cells (e.g., keratocytes in an in vitro toxicity assay). In this context, cellular toxicity can be measured by direct cell counts, trypan blue exclusion, or various metabolic activity studies such as 3 H-thymidine incorporation, as known to those skilled in the art.